Project Summary The CRISPR/Cas9 system has been developed in recent years for genome editing, and it has been rapidly and widely adopted by the scientific community. The main objective of this grant application is to develop next-generation single guide RNA (sgRNA), which is a critical component for CRISPR/Cas9 knockout assays. The sgRNA determines both the efficacy and specificity of CRISPR/Cas9 editing activities. However, the efficacy of sgRNA has not been adequately considered previously. The design of potent sgRNAs is highly desired, as inefficient genome editing by CRISPR/Cas9 will inevitably lead to significant waste of resources at the experimental validation stage. To address this challenge, we have developed both bioinformatics and experimental methods for improving the design of sgRNAs with high potency and specificity. Based on these newly established methods, we propose to further develop a new sgRNA design method to significantly improve the potency of CRIPSR/Cas9 knockout assays. Our new genome-wide sgRNA assays will lay a solid foundation for further commercial development of CRISPR/Cas9 products that can be used in a variety of gene knockout applications, such as the analysis of individual gene function, high-throughput functional screen and gene therapy.